Nearly two-thirds of all American adults are obese or overweight (1), resulting in over $93 billion in medical expenditures each year (2). Despite the growing obesity problem, the physiological regulation of energy balance is remarkably accurate resulting in the matching of caloric intake to caloric expenditure (3). This precision is dependent upon the body's ability to respond to internal cues relaying information about both long-term and short-term energy availability. The brain regulates energy balance in response to nutrient and hormonal cues, and neuronal fatty acid metabolism appears to be crucial to this mechanism. Malonyl-CoA is the building block for fatty acid synthesis and is abundant when acetyl-CoA accumulates. Multiple anorectic responses require malonyl-CoA, suggesting a role for the molecule as a nexus between fuel sensing and hormonal signaling in the hypothalamus. However, the key downstream actions of malonyl- CoA in key hypothalamic neurons have yet to be defined. The experiments outlined in this grant application are aimed to test the central hypothesis that malonyl-CoA acts to decrease food intake by stimulating the mammalian Target of Rapamycin (mTOR) in key hypothalamic nuclei. Recently, hypothalamic mTOR activation has been proposed as a critical hypothalamic fuel sensor (10). Both malonyl-CoA and mTOR are required for the dramatic anorectic effects of C75 (5), leptin (10), and CNTF (11). How these substrates might act together to alter ingestive behavior is unknown and represents a key overarching goal of the present proposal. Specific Aim 1: To demonstrate that elevated hypothalamic malonyl-CoA is required for stimulation of the mTOR pathway by anorectic agents. Specfic Aim 2: To test the hypothesis that elevated hypothalamic malonyl-CoA is sufficient to induce anorexia and activation of the mTOR pathway. Tests of these hypotheses will include behavioral and molecular assays during chronic pharmacologic inhibition of acetyl-CoA carboxylase (ACC), which is required for the production of malonyl-CoA. We will observe the effects of hypothalamic malonyl-CoA enrichment, achieved via knockdown of its degrading enzyme, malonyl-CoA decarboxylase (MCD) using intraarcuate infusion of an adeno-associated virus carrying a short hairpin RNA sequence targeted to the gene. PUBLIC HEALTH RELEVANCE: The proposed studies are expected to elucidate the role of malonyl-CoA in the regulation of food intake and to identify downstream targets linking the elevation of hypothalamic malonyl-CoA to the reduction of food intake. This contribution is significant, because an understanding of central fuel sensing will identify novel therapeutic strategies for the treatment and/or prevention of obesity.